Function Of Genes Regulated By C-Di-Gmp Bingding Protein Dgc3 In Bacillus Cereus 0-9

The second messenger cyclic di-GMP(c-di-GMP)controls the transition between motile and sessile lifestyles in the majority of bacteria by inducing biofilm formation and repressing motility.C-di-GMP also controls other cellular processes including virulence,cell-cell signaling,and cell cycle regulation.The synthesis of c-di-GMP is catalyzed by diguanylate cyclases(DGCs)containing the GGDEF domain,while its degradation is achieved through the phosphodiesterase activities of EAL and HD-GYP domains.C-di-GMP signals control bacteria adhesion on abiotic substances or eukaryotic cells and play important roles in lifestyle conversion of bacteria.Earlier studies in our laboratory revealed that a c-di-GMP binding protein Dgc3 existed in Bacillus cereus 0-9.Deletion of Dgc3 encoding gene results in biofilm production increase and swimming motility reduction.To investigate the effects of Dgc3 on cell process in B.cereus 0-9,Proteomic approaches were used to identify differentially expressed proteins between wild type and dgc3 gene deletion mutant and total of 112 proteins were identified.To explore the physiological functions of Dgc3 in B.cereus,seven differentially expression proteins encoding genes were selected to carry out functional analysis.Partially amino acid sequences of 7 proteins selected were analyzed by bioinformatics methods such as sequence alignment,protein domains analysis,signal peptide and transmembrane regions prediction.These proteins were identified respectively as heat shock protein DnaK,arginine deiminase AdI,2-oxoisovalerate dehydrogenase subunit alpha OgdH1,acetoacetyl-CoA reductase PhaB,metalloprotease InhAl,metalloprotease InhA2and a conserved hypothetical protein SmD.Gene knockout vectors were constructed through amplifying DNA fragment flanking open reading framework of 7 genes followed by linking it into shuttle plasmid pMAD which contained heat sensitive replicon.Seven single gene deletion mutants of B.cereus 0-9 were acquired through transformation of gene knockout plasmid into B.cereus component cells followed by screen erythromycin resistance colonies,homologous gene double crossover and DNA sequencing.Phenotypes,including growth,motility,biofilm formation,sporulation,exoprotease secretion,heat sensitivity,hemolytic activity and exopolysaccharides production,were assayed among wild type and seven single gene deletion mutants.The results revealed that loss of heat shock protein encoding gene dnaK in wild type 0-9 cause phenotype changeable significantly.The traits of dnaK mutant,compared to wild type of B.cereus 0-9,includes defective in motility and biofilm formation,reduction in hemolytic activity and heat sensitivity,lack in sporulation and increase in exopolysaccharides production.Other 6 single mutants displayed no obvious phenotype change under our tested conditions except mutant of smD which displayed swimming motility reduction.The results indicated heat shock protein DnaK played pleiotropic effects on cell process in B.cereus.Heat shock protein DnaK,a conserved protein existed in various bacteria,which contributes to maintain cell homeostasis by acting as a molecular chaperone.To reveal the functions of DnaK in B.cereus,fluorogenic quantitative PCR was used to assay the expression of dnaK gene in wild type 0-9 under high temperature conditions.The results indicated heat treatment to bacteria contributed to expression of dnaK.Heterogenous gene of dnaK from Bacillus subtilis 168 was used to carry out gene complementation to dnaK mutant of 0-9,which showed the complementation strain could restore partially defect phenotype.